Ebook Textbook of biochemistry (7th edition): Part 2

CHAPTER 28
Plasma Proteins
Chapter at a Glance
The reader will be able to answer questions on the following topics:
¾¾Plasma proteins
¾¾Electrophoresis
¾¾Albumin, functions, clinical significance
¾¾Hypoalbuminemia
¾¾Globulins, alpha, beta, gamma

Total blood volume is about 4.5 to 5 liters in adult
human being. If blood is mixed with an anticoagulant
and centrifuged, the cell components (RBC and WBC)
are precipitated. The supernatant is called plasma. About
55–60% of blood is made up of plasma.
i. If blood is withdrawn without anticoagulant and
allowed to clot, after about 2 hours liquid portion is
separated from the clot. This defibrinated plasma
is called serum, which lacks coagulation factors
including prothrombin and fibrinogen.
ii. Total protein content of normal plasma is 6 to 8 g/100
mL.
iii. The plasma proteins consist of albumin (3.5 to 5 g/dL),
globulins (2.5 – 3.5 g/dL) and fibrinogen (200– 400
mg/dL). The albumin : globulin ratio is usually between
1.2:1 to 1.5:1.
iv. Almost all plasma proteins, except immunoglobulins
are synthesized in liver. Plasma proteins are generally
synthesized on membrane-bound polyribosomes. Most
plasma proteins are glycoproteins.

¾¾Transport proteins in blood
¾¾Acute phase proteins in blood
¾¾Ceruloplasmin
¾¾Alpha-1 antitrypsin
¾¾Clotting factors

v. In laboratory, separation can be done by salts. Thus,
fibrinogen is precipitated by 10% and globulins by 22%
concentration of sodium sulfate. Ammonium sulfate
will precipitate globulins at half saturation and albumin
at full saturation.
vi. In clinical laboratory, total proteins in serum or plasma
of patients are estimated by Biuret method (see
Chapter 4). Albumin is quantitated by Bromo cresol
green (BCG) method, in which the dye is preferentially
bound with albumin, and the color is estimated
colorimetrically.

ELECTROPHORESIS
In clinical laboratory, electrophoresis is employed regularly
for separation of serum proteins. The term electrophoresis
refers to the movement of charged particles through
an electrolyte when subjected to an electric field. The
details are given in Chapter 35. Normal and abnormal
electrophoretic patterns are shown in Figures 28.1 and 28.2.


Chapter 28:  Plasma Proteins

379

Normal Patterns and Interpretations

Abnormal Patterns in Clinical Diseases

i. In agar gel electro­phoresis, normal serum is separated
into 5 bands. Their relative concentrations are given
below:
Albumin
: 55–65%
Alpha-1 globulin : 2–4%
Alpha-2 globulin : 6–12%
Beta globulin
: 8–12%
Gamma globulin : 12–22%
ii. Albumin has the maximum and gamma globulin
has the minimum mobility in the electrical field.
iii. G a m m a g l o b u l i n s c o n t a i n t h e a n t i b o d i e s
(immunoglobulins). Most of the alpha-1 fraction is
made up of alpha-1 antitrypsin. Alpha-2 band is mainly
made up by alpha-2 macroglobulin. Beta fraction
contains low density lipoproteins.

Various abnormalities can be identified in the electrophoretic
pattern (Figs 28.1A and B).
1. Chronic infections: The gamma globulins are
increased, but the increase is smooth and widebased.
2. Multiple myeloma: In para-proteinemias, a sharp
spike is noted and is termed as M-band. This is due
to monoclonal origin of immunoglobulins in multiple

myeloma (Fig. 28.2).
3. Fibrinogen: Instead of serum, if plasma is used for
electrophoresis, the fibrinogen will form a prominent
band in the gamma region, which may be confused with
the M-band.
4. Primary immune deficiency: The gamma globulin
fraction is reduced.
5. Nephrotic syndrome: All proteins except very big
molecules are lost through urine, and so alpha-2
fraction (containing macroglobulin) will be very
prominent.
6. Cirrhosis of liver: Albumin synthesis by liver is
decreased, with a compensatory excess synthesis of
globulins by reticuloendothelial system. So albumin
band will be thin, with a wide beta fraction; sometimes
beta and gamma fractions are fused.

Fig. 28.1A: Serum electrophoretic patterns. 1 = Normal pattern; 2 =

Multiple myeloma (M band) between b and g region; 3 =Chronic
infection, broad based increase in g region; general increase in
a1 and a2 bands; 4 = Nephrotic syndrome; hypoalbuminemia;
prominent a2 band; 5 = Cirrhosis of liver; decreased albumin;
6 = Plasma showing fibrinogen (normal condition). This may be
mistaken for paraproteins

Fig. 28.1B: Serum electrophoretic patterns


380 Textbook of Biochemistry

7. Chronic lymphatic leukemia, gamma globulin
fraction is reduced.
8. Alpha-1 antitrypsin deficiency: The alpha-1 band is
thin or even missing.

ALBUMIN
i. The name is derived from the white precipitate formed
when egg is boiled (Latin, albus = white). Albumin
constitutes the major part of plasma proteins.
ii. It has one polypeptide chain with 585 amino acids. It
has a molecular weight of 69,000 D. It is elliptical in
shape.
iii. It is synthesized by hepatocytes; therefore, estimation
of albumin is a liver function test (see Chapter 26).
Albumin is synthesized as a precursor, and the signal
peptide is removed as it passes through endoplasmic
reticulum.
iv. Albumin can come out of vascular compartment. So
albumin is present in CSF and interstitial fluid.
v. Half-life of albumin is about 20 days. Liver produces
about 12 g of albumin per day, representing about 25%
of total hepatic protein synthesis.
Half-life: Each plasma protein has a characteristic half-life in circulation;
e.g. half-life of albumin is 20 days, and that of haptoglobin is 5 days.
The half-life is studied by labeling the pure protein with radioactive
chromium (51Cr). A known quantity of the labeled protein is injected into
a normal person, and blood samples are taken at different time intervals.
Half-life of a protein in circulation may be drastically reduced when
proteins are lost in conditions, such as Crohn's disease (regional ileitis)
or protein losing enteropathy.

Fig. 28.2: Normal and abnormal electrophoretic patterns

Functions of Albumin
Colloid Osmotic Pressure of Plasma
i. The total osmolality of serum is 278–305 mosmol/kg
(about 5000 mm of Hg). But this is produced mainly
by salts, which can pass easily from intravascular to
extravascular space. Therefore, the osmotic pressure
exerted by electrolytes inside and outside the vascular
compartments will cancel each other. But proteins
cannot easily escape out of blood vessels, and therefore,
proteins exert the ‘effective osmotic pressure'. It
is about 25 mm Hg, and 80% of it is contributed
by albumin. The maintenance of blood volume is
dependent on this effective osmotic pressure.
ii. According to Starling's hypothesis, at the capillary
end, the blood pressure (BP) or hydrostatic pressure
expels water out, and effective osmotic pressure (EOP)
takes water into the vascular compartment (Fig. 28.3).
iii. At arterial end of the capillary, BP is 35 mm Hg and
EOP is 25 mm; thus water is expelled by a pressure of
10 mm Hg. At the venous end of the capillary, EOP is
25 mm and BP is 15 mm, and therefore water is imbibed
with a pressure of 10 mm. Thus, the number of water
molecules escaping out at arterial side will be exactly
equal to those returned at the venous side and therefore
blood volume remains the same.
iv. If protein concentration in serum is reduced, the EOP
is correspondingly decreased. Then return of water into

blood vessels is diminished, leading to accumulation
of water in tissues. This is called edema.


Chapter 28:  Plasma Proteins

381

v. Edema is seen in conditions where albumin level in
blood is less than 2 g/dL (see hypoalbuminemia).

Clinical Applications

Transport Function

Albumin-fatty acid complex cannot cross blood-brain barrier
and hence fatty acids cannot be taken up by brain. The
bilirubin from albumin may be competitively replaced
by drugs like aspirin. Being lipophilic, unconjugated
bilirubin can cross the blood brain barrier and get deposited
in brain. The brain of young children are susceptible; free
bilirubin deposited in brain leads to kernicterus and mental
retardation (see Chapter 21).

Albumin is the carrier of various hydrophobic substances in
the blood. Being a watery medium, blood cannot solubilize
lipid components.
i. Bilirubin and non-esterified fatty acids are specifically
transported by albumin.
ii. Drugs (sulfa, aspirin, salicylate, dicoumarol,

phenytoin).
iii. Hormones: Steroid hormones, thyroxine.
iv. Metals: Albumin transports copper. Calcium and heavy
metals are non-specifically carried by albumin. Only
the unbound fraction of drugs is biologically active.

Buffering Action
All proteins have buffering capacity. Because of its high
concentration in blood, albumin has maximum buffering
capacity (see Chapter 29). Albumin has a total of 16 histidine
residues which contribute to this buffering action.

Nutritional Function
All tissue cells can take up albumin by pinocytosis. It is
then broken down to amino acid level. So albumin may be
considered as the transport form of essential amino acids
from liver to extrahepatic cells. Human albumin is clinically
useful in treatment of liver diseases, hemorrhage, shock
and burns.

Blood Brain Barrier

Drug Interactions
When two drugs having high affinity to albumin are
administered together, there may be competition for the
available sites, with conse­quent displacement of one drug.
Such an effect may lead to clinically significant drug
interactions, e.g. phenytoin-dicoumarol interaction.

Protein-bound Calcium

Calcium level in blood is lowered in hypoalbuminemia.
Thus, even though total calcium level in blood is lowered,
ionized calcium level may be normal, and so tetany may not
occur (see Chapter 39). Calcium is lowered by 0.8 mg/dL
for a fall of 1 g/dL of Albumin.

Therapeutic Use
Human albumin is therapeutically useful to treat burns,
hemorrhage and shock.

Edema
Hypoalbuminemia will result in tissue edema (see Starling's
law).

Fig. 28.3: Starling hypothesis

a. Malnutrition, where albumin synthesis is depressed
(generalized edema)
b. Nephrotic syndrome, where albumin is lost through
urine (facial edema)
c. Cirrhosis of liver (mainly ascites), where albumin
synthesis is less and it escapes into ascitic fluid
d. Chronic congestive cardiac failure: Venous congestion
will cause increased hydrostatic pressure and decreased
return of water into capillaries and so pitting edema of
feet may result.


382 Textbook of Biochemistry
Normal Value

Chronic Infections

Normal level of Albumin is 3.5–5 g/dL. Lowered level
of albumin (hypoalbuminemia) has important clinical
significance.

Gamma globulins are increased, but the increase is smooth
and wide based (Fig. 28.1A).

Hypoalbuminemia
a. Cirrhosis of liver: Synthesis is decreased.
b. Malnutrition: Availability of amino acids is reduced
and albumin synthesis is affected.
c. Nephrotic syndrome: Permeability of kidney
glomerular membrane is defective, so that albumin is
excreted in large quantities.
d. Albuminuria: Presence of albumin in urine is called
albuminuria. It is always pathological. Large quantities
(a few grams per day) of albumin is lost in urine in
nephrotic syndrome. Small quantities are lost in urine
in acute nephritis, and other inflammatory conditions
of urinary tract. Detection of albumin in urine is
done by heat and acetic acid test (see Chapter 27).
In microalbuminuria or minimal albuminuria or
paucialbuminuria, small quantity of albumin (30–300
mg/d) is seen in urine (Paucity = small in quantity).
e. Protein losing enteropathy : Large quantities of
albumin is lost from intestinal tract.
f. Analbuminemia is a very rare condition, where

defective mutation in the gene is responsible for
absence of synthesis.

Albumin-Globulin Ratio
In hypoalbuminemia, there will be a compensatory increase
in globulins which are synthesized by the reticuloendo­thelial
system. Albumin-globulin ratio (A/G ratio) is thus altered
or even reversed. This again leads to edema.

Hypoproteinemia
Since albumin is the major protein present in the blood, any
condition causing lowering of albumin will lead to reduced
total proteins in blood (hypoproteinemia).

Hypergammaglobulinemias
Low Albumin Level
When albumin level is decreased, body tries to compensate
by increasing the production of globulins from reticuloendothelial system.

Multiple Myeloma
Drastic increase in globulins are seen in para-proteinemias,
when a sharp spike is noted in electrophoresis. This is
termed as M-band because of the monoclonal origin of
immunoglobulins (Figs 28.1B and 28.2). The monoclonal
origin of immunoglobulins is seen in multiple myeloma (see
Chapter 55). Monoclonal gammopathies are characterized
by the presence of a monoclonal protein, which can be
detected by serum protein electrophoresis and typed by
immunofixation electrophoresis.The light chains are produced
in excess which is excreted in urine as Bence Jones proteins

(BJP) when their serum level increases. Multiple myeloma is
the most common type of monoclonal gammopathy. Free light
chain assay along with kappa and lambda ratio in serum and
urine is found to be very useful in early diagnosis, monitoring
the response to treatment and prediction of prognosis.

TRANSPORT PROTEINS
Blood is a watery medium; so lipids and lipid soluble
substances will not easily dissolve in the aqueous medium
of blood. Hence such molecules are carried by specific
carrier proteins. Their important features are summarized
in Table 28.1.
1. Albumin : It is an important transport protein, which
carries bilirubin, free fatty acids, calcium and drugs (see
above).
2. Pre-albumin or Transthyretin: It is so named because
of its faster mobility in electrophoresis than albumin.
It is more appropriately named as Transthyretin or
Thyroxin binding pre-albumin (TBPA), because it
carries thyroid hormones, thyroxin (T4) and tri-iodo
thyronine (T3). Its half-life in plasma is only 1 day.
3. Retinol binding protein (RBP) : It carries vitamin A
(see Chapter 36). It is a low molecular weight protein,
and so is liable to be lost in urine. To prevent this loss,
RBP is attached to pre-albumin; the complex is large
and will not pass through kidney glomeruli. It is a
negative acute phase protein.
4. Thyroxine binding globulin (TBG) : It is the specific
carrier molecule for thyroxine and tri-iodo thyronine.
TBG level is increased in pregnancy; but decreased in

nephrotic syndrome.


Chapter 28:  Plasma Proteins

383

TABLE 28.1: Carrier proteins or transport proteins of plasma
Name

Plasma
level

Molecular
wt (Dalton )

Compound
bound or
transported

Electrophoretic
mobility

Biological and clinical significance

Albumin

3.5–5 g/dL

69,000

Fatty acids,
bilirubin,
calcium,
thyroxine,
heavy metals,
drugs
e.g. aspirin, sulfa

Maximum
anodal
migration

Bilirubin competes with aspirin for binding
sites on albumin

Prealbumin
(Transthyretin)

25–30 mg/
dL

54,000

Steroid
hormones,
thyroxine,
retinol

Faster than

albumin

Rich in tryptophan. Half-life is 1day
It is a negative acute phase protein
Transports T3 and T4 losely

Retinol
binding
protein (RBP)

3–6 mg/dL

21,000

Retinol
(Vitamin A)

a1

Synthesized by liver. RBP has a short
half-life. Level indicates vitamin A status
Useful to assess the protein turn over rate

Thyroxine
binding
globulin (TBG)

1–2 mg/dL

58,000

Thyroxine

a1

Assessment of the binding sites on TBG
is important in studying thyroid function
It is synthesized in liver

Transcortin;
Cortisol
binding
globulin (CBG)

3–3.5 mg/
dL

52,000

Cortisol
and
Corticosterone

a1

Synthesized by liver. Increased in
pregnancy. Free unbound fraction of
hormone is biologically active

Haptoglobin

(Hp)

40-175 mg/ 100,000
dL
to
400,000

Hemoglobin

a2

Synthesized in liver. Low level indicates
hemolysis. Half-life of Hp is 5 days; but
that of Hb-Hp is only 90 minutes. It is an
acute phase protein (see Chapter 35)

Transferrin

200–300
mg/dL

76,500

Iron 33%
saturated

b

Conserves iron by preventing iron loss
through urine (see Chapter 35)

Hemopexin

50–100
mg/dL

57,000

Free heme

b

Helps in preventing loss of heme (and so
iron also) from body (see Chapter 35)

5. Transcortin: It is also known as Cortisol binding
globulin (CBG). It is the transport protein for cortisol
and corticosterone.
6. Haptoglobin: Haptoglobin (for hemoglobin),
Hemopexin (for heme) and Transferrin (for iron) are
important to prevent loss of iron from body.

Polymorphism
The term polymorphism is applied when the protein exists
in different phenotypes in the population; but only one form
is seen in a particular person. Haptoglobin, transferrin,
ceruloplasmin, alpha-1-antitrypsin and immunoglobulins
exhibit polymorphism. For example, Haptoglobin (Hp)
exists in three forms, Hp1–1, Hp2–1, and Hp2–2. Two genes,
designated Hp1 and Hp2 are responsible for these polymorphic

forms. Their functional capabilities are the same. These
polymorphic forms are recognized by electrophoresis or by

immunological analysis. Study of polymorphism is useful
for genetic and anthropological studies.

ACUTE PHASE PROTEINS
The level of certain proteins in blood may increase 50
to 1000 folds in various inflammatory and neoplastic
conditions. Such proteins are acute phase proteins. Important
acute phase proteins are described below:

C-Reactive Protein (CRP)
So named because it reacts with C-polysaccharide of
capsule of pneumococci. CRP is a beta-globulin and has
a molecular weight of 115–140 kD. It is synthesized in
liver. It can stimulate complement activity and macrophage
phagocytosis. When the inflammation has subsided, CRP
quickly falls, followed by ESR (erythrocyte sedimentation
rate). CRP level, especially high sensitivity C-reactive


384 Textbook of Biochemistry
protein level in blood has a positive correlation in predicting
the risk of coronary artery diseases (see Chapter 25).

Ceruloplasmin
i. Ceruloplasmin is blue in color (Latin, caeruleus=blue).
It is an alpha-2 globulin with molecular weight of
160,000 Daltons. It contains 6 to 8 copper atoms per

molecule.
ii. Ceruloplasmin is mainly synthesized by the hepatic
parenchymal cells and a small portion by lymphocytes
and macrophages. After the formation of peptide part
(apo-Cp) copper is added by an intracellular ATPase
and carbohydrate side chains are added to make it a
glycoprotein (holo-Cp). The normal plasma half-life
of holo-Cp is 4–5 days.
iii. Ceruloplasmin is also called Ferroxidase, an enzyme
which helps in the incorporation of iron into transferrin
(see Chapter 39). It is an important antioxidant in
plasma.
iv. About 90% of copper content of plasma is bound
with ceruloplasmin, and 10% with albumin. Copper is
bound with albumin loosely, and so easily exchanged
with tissues. Hence, transport protein for copper is
Albumin.
v. Lowered level of ceruloplasmin is seen in Wilson's
disease, malnutrition, nephrosis, and cirrhosis.
vi. Ceruloplasmin is an acute phase protein. Increased
plasma Cp levels are seen in active hepatitis, biliary
cirrhosis, hemochromatosis, and obstructive biliary
disease, pregnancy, estrogen therapy, inflammatory
conditions, collagen disorders and in malignancies.
Drugs increasing the ceruloplasmin level are, estrogen
and contraceptives.
Reference blood levels of ceruloplas­min are:
Adults
Males
22 – 40 mg/dL

Females25 – 60 mg/dL
Pregnancy30 – 120 mg/dL

Wilson's Disease
a. Level is reduced to less than 20 mg/dL in Wilson's
hepa­to­­lenticular degeneration. It is an inheri­ted
autosomal recessive condition. Incidence of the disease
is 1 in 50,000.
b. The basic defect is a mutation in a gene encoding a
copper binding ATPase in cells, which is required

for excretion of copper from cells. So, copper is not
excreted through bile, and hence copper toxicity. Please
also see Chapter 39, under copper metabolism.
c. Increased copper content in hepatocyte inhibits the
incorporation of copper to apo-ceruloplasmin. So
ceruloplasmin level in blood is decreased.

Clinical Features
a. Accumulation in liver leads to hepatocellular degeneration
and cirrhosis.
b. Deposits in brain basal ganglia leads to lenticular
degeneration and neurological symptoms.
c. Copper deposits as green or golden pigmented ring
around cornea; this is called Kayser-Fleischer ring.
d. Treatment consists of a diet containing low copper and
injection of D-penicillamine, which excretes copper
through urine. Since zinc decreases copper absorption,
zinc is useful in therapy.

Alpha-1 Antitrypsin (AAT)
It is otherwise called alpha-anti-proteinase or protease
inhibitor. It inhibits all serine proteases (proteolytic
enzymes having a serine at their active center), such as
plasmin, thrombin, trypsin, chymotrypsin, elastase, and
cathepsin. Serine protease inhibitors are abbreviated as
Serpins.

The AAT is synthesized in liver. It is a glycoprotein with
a molecular weight of 50 KD. It forms the bulk of molecules
in serum having alpha-1 mobility. Normal serum level
is 75 – 200 mg/dL. AAT deficiency causes the following
conditions:
Emphysema: The incidence of AAT deficiency is 1 in 1000
in Europe, but uncommon in Asia. The total activity of AAT
is reduced in these individuals. Bacterial infections in lung
attract macrophages which release elastase. In the AAT
deficiency, unopposed action of elastase will cause damage to
lung tissue, leading to emphysema. About 5% of emphysema
cases are due to AAT deficiency.
Nephrotic syndrome: AAT molecules are lost in urine, and
so AAT deficiency is produced.
Liver Cirrhosis
Deficiency of a1 antitrypsin is the most common genetic cause for liver
disease in infants and children. It starts as “neonatal hepatitis syndrome”
and may progress to liver failure and cirrhosis. a1 antitrypsin can be
detected by serum electrophoresis.

Chapter 28:  Plasma Proteins
Alpha-2 Macroglobulin (AMG)
It is a tetrameric protein with molecular weight of 725 KD. It is the
major component of alpha-2 globulins. It is synthesized by hepatocytes
and macrophages. AMG inactivates all proteases, and is an important in
vivo anti-coagulant. AMG is the carrier of many growth factors, such as
platelet derived growth factor (PDGF). Normal serum level is 130–300 mg/
dL. Its concentration is markedly increased (up to 2 –3 g/dL) in Nephrotic
syndrome, where other proteins are lost through urine.

Negative Acute Phase Proteins
During an inflammatory response, some proteins are seen
to be decreased in blood; those are called negative acute
phase proteins. Examples are albumin, transthyretin (prealbumin), retinol binding protein and transferrin.
Transferrin is a specific iron binding protein (see
Chapter 39). It has a half-life of 7–10 days and is used as a
better index of protein turnover than albumin.
Plasma contains many enzymes (see Chapter 23),
protein hormones (see Chapter 50) and immunoglobulins
(see Chapter 55). A comprehensive list of normal values for
the substances present in blood is given in the Appendix II.

CLOTTING FACTORS
The word coagulation is derived from the Greek term,
"coagulare" = to curdle. The biochemical mechanism of
clotting is a typical example of cascade activation.
The coagulation factors are present in circulation as
inactive zymogen forms. They are converted to their active
forms only when the clotting process is initiated. This would
prevent unnecessary intravascular coagulation. Activation

process leads to a cascade amplification effect, in which
one molecule of preceding factor activates 1000 molecules
of the next factor. Thus within seconds, a large number
of molecules of final factors are activated. The clotting
process is schematically represented in Figure 28.4 and the
characteristics of coagulation factors are shown in Table 28.2.
Several of these factors require calcium for their
activation. The calcium ions are chelated by the gamma
carboxyl group of glutamic acid residues of the factors,
prothrombin, VII, IX, X, XI and XII. The gamma
carboxylation of glutamic acid residues is dependent on
vitamin K (see Chapter 33), and occurs after synthesis of
the protein (post-translational modification).

Prothrombin
It is a single chain zymogen with a molecular weight of
69,000 D. The plasma concentration is 10–15 mg/dL. The

385

prothrombin is converted to thrombin by Factor Xa, by the
removal of N-terminal fragment.

Thrombin
It is a serine protease with molecular weight of 34,000 D.
The Ca++ binding of prothrombin is essential for anchoring
the prothrombin on the surface of platelets. When the
terminal fragment is cleaved off, the calcium binding sites
are removed and so, thrombin is released from the platelet
surface.

Fibrinogen
The conversion of fibrinogen to fibrin occurs by cleaving
of Arg-Gly peptide bonds of fibrinogen. Fibrinogen has a
molecular weight of 340,000 D and is synthesised by the
liver. Normal fibrinogen level in blood is 200–400 mg/
dL. The fibrin monomers formed are insoluble. They align
themselves lengthwise, aggregate and precipitate to form
the clot. Fibrinogen is an acute phase protein.
Fibrinolysis
Unwanted fibrin clots are continuously dissolved in vivo by Plasmin,
a serine protease. Its inactive precursor is plasminogen (90 kD). It is
cleaved into two parts to produce the active plasmin. Plasmin in turn, is
inactivated by alpha-2 antiplasmin.
Tissue plasminogen activator (TPA) is a serine protease present
in vascular endothelium. TPA is released during injury and then binds to
fibrin clots. Then TPA cleaves plasminogen to generate plasmin, which
dissolves the clots.

Urokinase is another activator of plasminogen. Urokinase is so
named because it was first isolated from urine. Urokinase is produced by
macrophages, monocytes and fibroblasts. Streptokinase, isolated from
streptococci is another fibrinolytic agent.

Clinical Significance
Thrombosis in coronary artery is the major cause of
myocardial infarction (heart attack). If TPA, urokinase or
streptokinase is injected intravenously in the early phase
of thrombosis, the clot may be dissolved and recovery of
patient is possible.

Prothrombin Time (PT)
It evaluates the extrinsic coagulation pathway, so that if any of the
factors synthesized by the liver (factors I, II, V, VII,IX and X) is deficient
prothrombin time will be prolonged. It is the time required for the clotting
of whole blood (citrated or oxalated) after addition of calcium and tissue
thromboplastin. So, fibrinogen is polymerized to fibrin by thrombin.

It is commonly assessed by the “one stage prothrombin time of Quick”
(named after the inventor). The results are expressed either in seconds or as
a ratio of the plasma prothrombin time to a control plasma time. The normal


386 Textbook of Biochemistry
control PT is 9 – 11 seconds. A prolongation of 2 seconds is considered as
abnormal. Values more than 14 seconds indicate impending hemorrhage.
The PT is prolonged if any of the concerned factors are deficient. The present
techniques express the prothrombin level as a ratio as INR (Internationalized
ratio).

Liver dysfunction of acute onset will be reflected as prolonged
prothrombin time. Out of 13 clotting factors, 11 are synthesized by
the liver. Their synthesis is dependent on availability of vitamin K and
normal hepatocellular function.

Prolonged PT may be the initial supportive laboratory parameter to
diagnose an acute liver disease. Persistent and progressing prolonged PT
is suggestive of fulminant liver failure.

PT measurements are useful to differentiate cholestasis and severe
hepatocellular disease. When prolonged PT result is obtained; give

vitamin K by intramuscular injection and after 4 hours recheck PT.
If the PT becomes normal after vitamin K injection (which is needed
for post-translational modification of prothrombin) the diagnosis of
cholestasis can be made. If the PT is prolonged, the possibility is severe
hepatocellular disease.

ABNORMALITIES IN COAGULATION
Hemophilia A (Classical Hemophilia)
This is an inherited X-linked recessive disease affecting
males and transmitted by females. Male children of
hemophilia patients are not affected; but female children will
be carriers, who transmit the disease to their male offspring

(Fig. 28.5). This is due to the deficiency of factor VIII (anti
hemophilic globulin) (AHG). It is the commonest of the
inherited coagulation defects.

There will be prolongation of clotting time. Hence, even
trivial wounds, such as tooth extraction will cause excessive
loss of blood. Patients are prone to internal bleeding into
joints and intestinal tract.

Until recently the treatment consisted of administration
of AHG, prepared from pooled sera every 3 months. Since
this was not generally available, the usual treatment was to
transfuse blood periodically, which may lead to eventual
iron overload, hemochromatosis (see Chapter 39). Several
hemophilia patients, receiving repeated transfusions became
innocent victims of AIDS. Pure AHG is now being produced
by recombinant technology and is the treatment of choice.

Hemophilia B or Christmas Disease
It is due to factor IX deficiency. The Christmas disease is named after
the first patient reported with this disease. Similar deficiencies of factors
X and XI are also reported. In these diseases, the manifestations will be
similar to classical hemophilia.

Other Disorders
Acquired hypofibrinogenemia or afibrinogenemia may occur as a
complication of premature separation of placenta or abruptio placenta.

TABLE 28.2: Factors involved in coagulation process
No.

Name

Molecular
weight
(Daltons)

Electrophoretic
mobility

Activated by

Function

I

Fibrinogen

340,000

b and g

Thrombin

Forms the clot (fibrin)

II

Prothrombin

69,000

a2

Factor Xa

Activation of fibrinogen and factors XIII, VIII
and V

IV

Calcium

—

Activation of factor II, VII, IX, X,XI and XII

V

Labile factor

200,000

Thrombin

Binding of prothrombin to platelet

VII

Proconvertin; serum
prothrombin convertin
antecedent (SPCA)

45,500

Thrombin

Activation of factor X

VIII

Antihemophilic globulin (AHG)

1,200,000

b2

Thrombin

Activation of factor X

IX

Plasma thromboplastincomponent (PTC);
Christmas factor

62,000

a

Factor Xla

Activation of factor X

X

Stuart Prower factor

59,000

a

Factor IXa

Activation of prothrombin

XI

Plasma thromboplastin
anticedent (PTA)

200,000

bg

Factor XIIa

Activation of factor IX

XII

Hageman factor

80,000

Kallikrein

Activation of factor XI

XIII

Fibrin stabilizing factor (Liki
Lorand factor)

320,000

Thrombin

Stabilization of fibrin clot by forming cross
links

Prekallikrein

85,000

bg

g

Activation of factor XII


Chapter 28:  Plasma Proteins
Proteolytic thromboplastic substances may enter from placenta to
maternal circulation which set off the clotting cascade (disseminated
intravascular coagulation or DIC). But the clots are usually degraded
immediately by plasminolysis. Continuation of this process leads to
removal of all available prothrombin and fibrinogen molecules leading
to profuse postpartum hemorrhage.

In some cases of carcinoma of pancreas, trypsin is released into
circulation leading to intravascular coagulation. This may be manifested
as fleeting thrombophlebitis. Trousseau diagnosed his own fatal
disease as cancer of pancreas when he developed thrombophlebitis. The
combi­nation of carcinoma of pancreas, migratory thrombophlebitis and
consumption coagulopathy is termed as Trousseau's triad.

Prothrombin G20210A Polymorphism

Another hereditary thrombophilia, the G20210A polymorphism in the
prothrombin gene elevates the plasma concentrations of prothrombin
(FII) without changing the amino acid sequence of the protein. Patients

Fig. 28.4: Cascade pathway of coagulation

387

with this mutation have PT and aPTT results that fall within the normal
range, as well as normal functional clot-based studies. DNA studies will
show a G-to-A substitution in the 3’-untranslated region of prothrombin
gene at nucleotide 20210.

Protein C and S Deficiency
These two vitamin K-dependent factors interrupt the activity of clotting
factors V and VIII. Activated protein C is a proteolytic enzyme, while
protein S is an essential co-factor.

Antithrombin Deficiency
AT, formerly called AT III, is a vitamin K-independent glycoprotein that
is a major inhibitor of thrombin and other coagulation serine proteases,
including factors Xa and IXa. AT forms a competitive 1:1 complex with its
target but only in the presence of a negatively charged glycosaminoglycan,
such as heparin or heparin sulfate. Patients with AT deficiency will have
little AT III activity as measured in a chromogenic assay.


388 Textbook of Biochemistry
Anticoagulants
They are mainly two types: 1. Acting in vitro to prevent coagulation of

collected blood and 2. Acting in vivo to prevent and regulate coagulation.

The first group of anticoagulant removes calcium which is essential
for several steps on clotting. Oxalates, citrate and EDTA belong to this
group.

Heparin and antithrombin III are the major in vivo anticoagulants.
The heparin-antithrombin complex exerts an inhibitory effect on the serine
protease which activates the clotting factors. Alpha-2 macroglobulin has
anticoagulant activity.

Heparin is also used as an anticoagulant for in vitro system, e.g. in
dialysis and in thromboembolic diseases. It is also used in the treatment
of intravascular thrombosis. Since vitamin K is essential for coagulation,
antagonists to vitamin K are used as anticoagulants, especially for
therapeutic purposes, e.g. Dicoumarol and Warfarin (see Chapter 36,
under Vitamin K).

Coagulation Tests
Laboratory tests for hemostasis typically require citrated plasma derived
from whole blood. Specimens should be collected into tubes containing
3.2% sodium citrate (109 mM) at a ratio of 9 parts blood and 1 part
anticoagulant. The purpose of the citrate is to remove calcium ions that
are essential for blood coagulation (Table 28.3).

Antiphospholipid Syndrome (APS)
It is frequently associated with a markedly prolonged aPTT, leading
to a concern that the affected individual might be at risk for a major
hemorrhage. Not only is this highly unlikely, but as a prothrombotic
state, APLS is typically associated with venous thromboembolism

and/or arterial thrombosis. The condition may also present with fetal
loss or stillbirth, which occurs as a result of placental inflammation or
thrombosis. Individuals with APLS have antibodies known as lupus
anticoagulants (LA). These antibodies are directed to complexes of beta2 glycoprotein I/phospholipid or prothrombin/phospholipid, and they
interfere with and prolong in vitro clotting assays. In the body’s vascular
system, however, the presence of endothelial cells and leukocytes, as
well as many other components that are absent from the simplified

Fig. 28.5: Inheritance pattern of hemophilia

in vitro clotting assay, increase the likelihood of clotting. The classic
laboratory findings in APLS patients are prolonged aPTT, and normal
PT. Adding excess phospholipid to the aPTT assay, however, reduces
the clotting time. This is the basis for the so-called LA assay. The APS
is an important cause of acquired thromboembolic complications and
pregnancy morbidity. APAs are also found in other autoimmune diseases,
in patients receiving drugs such as procainamide and chlorpromazine, in
patients with infections (HIV, hepatitis, malaria, and others), and also in
association with malignancy.

Clinical Case Study 28.1
A severe form of obstructive lung disease starting with
dyspnea and leading to emphysema was found in several
members of the same family. Blood analysis of the
surviving members of the family revealed abnormally
low concentration of a1 antitrypsin. What is the basis of
this condition?

Clinical Case Study 28.2
A male child, born to a normal young couple, was found to

develop hemorrhagic tendency quite early in life. History
revealed that the mother was the only daughter of a family who
did not have any male offspring during the past 2 generations.
A. What are the possible causes?
B. How will you explain the nature of inheritance?
C. What is the advice to be given to the parents regarding
bringing up the son and having another child.

Clinical Case Study 28.1 Answer
Emphysema, a lung disease characterized by destruction
of alveolar walls, has many causes including airway
infections, cigarette smoking, air pollution and hereditary
origin. Deficiency of a1 antitrypsin leads to development of
emphysema. a1 antitrypsin makes up most of the proteins
in a1 globulin band during serum protein electrophoresis.
Lungs contain a natural enzyme called neutrophil
elastase that digests damaged aging cells and bacteria and
promotes healing of lung tissue. Being non-specific it can
attack lung tissue itself; but a1 antitrypsin protects against
this process by destroying excess amount of this enzyme.
Absence of a1 antitrypsin can lead to destruction of lung
tissue and emphysema.
Clinical features of a1 antitrypsin deficiency include
shortness of breath, reduced exercise tolerance, wheezing,
recurrent respiratory infections and in advanced cases,
difficulty in breathing. Smoking exacerbates the condition.
About 10% of patients can have liver damage.


Chapter 28:  Plasma Proteins

389

TABLE 28.3: Assays for clotting factors
Name

Parameter measured

Prothromin time (PT)

Time in seconds taken for the patient's sample to clot; thromboplastin reagent is added

Partial thromboplastin time (PTT)

A measure of how well patient's blood will clot

Activated partial thromboplastin
time (aPTT)

Initiated by adding a negatively charged surface like silica to the plasma and a phoispholipid extract
that is free of tissue factor

Thrombin time (TT)

Assess hemostasis. Measures ability of fibrinogen to form fibrin strands in vitro. Exogenous thrombin
is added to pre-warmed plasma

D-Dimer

Assess hemostatic function. D-Dimer is formed by degradation of fibrin clots by thrombin, activated

factor XIII and plasmin. High level indicates increased risk of recurrent thromboembolism. High
negative predictive value of thrombosis

Activated clotting time

Whole blood is mixed with a clot activator. Normally takes 70 – 180 seconds. Bedside monitoring of
high dose heparin therapy

Anti-Xa test

Exogenous factor Xa and anti-thrombin (AT )in excess and chromogenic substrate fro Xa. Heparin
present complexes with AT and inactivates factor Xa. Any excess Xa will release the chromophore
from the substrate. Adjustment of patient’s heparin level

Coagulation factor assay

Determines the level of various coagulation factors. Factor deficient plasma is mixed with the
specific factor being tested by adding patient’s diluted citrated plasma. The patient’s specimen
supplies the missing factor and the assay is completed by performing a standard PT Calibrated by
using standard reference plasma

Diagnosis is by estimation of a1 antitrypsin levels,
arterial blood gas analysis, chest X-ray, CT scan of chest,
pulmonary function tests and genetic testing. Treatment
involves supplementation of a1 antitrypsin and antioxidants.

Clinical Case Study 28.2 Answer
Hemophilia (see description in this chapter).

QUICK LOOK OF CHAPTER 28

1. Total plasma protein content is 6–8 g/dL of which
albumin is 3.5–5 g/dL and the rest is globulin. Almost
all plasma proteins are synthesized in the liver except
immunoglobulins.
2. On agar gel electrophoresis albumin has maximum
mobility while gamma globulin has minimum
mobility.
3. In chronic infection, gamma globulins are increased
smoothly, while in paraproteinemias, M band is
seen. The alpha 2 fraction is increased in nephrotic
syndrome while albumin is decreased in liver cirrhosis,
malnutrition, nephrotic syndrome.

4. Albumin contributes to colloid osmotic pressure of
plasma, has buffering capacity and is a transport
medium for various hydrophobic substances.
5. Hyper gamma globulinemia is seen in conditions
of hypoalbuminemia, chronic infection and para
proteinemias.
The transport proteins in blood are albumin, prealbumin (transthyretin), RBP, TBG, transcortin and
haptoglobin.
6. Polymorphism is when the protein exists in different
phenotypes in the population, but only one form is
seen in a particular person. This is seen in haptoglobin,
transferrin, ceruloplasmin, alpha1 antitrypsin and
immunoglobulins.
7. The levels of certain proteins in blood may increase
50 –100-fold in various inflammatory and neoplastic
conditions. Such proteins are called acute phase proteins.
For example, CRP, ceruloplasmin, haptoglobins, alpha1

acid glycoprotein, alpha1 antitrypsin and fibrinogen.
8. Proteins that are decreased in blood during inflammatory
response are called negative acute phase proteins. For
example, albumin, transthyretin, transferrin.


CHAPTER 29
Acid-Base
Balance and pH
Chapter at a Glance
The reader will be able to answer questions on the following topics:
¾¾Acids and bases
¾¾pH
¾¾Buffers
¾¾Acid base balance in the body
¾¾Bicarbonate buffer system
¾¾Respiratory regulation of pH

Hydrogen ions (H+) are present in all body compartments.
Maintenance of appropriate concentration of hydrogen ion
(H+) is critical to normal cellular function. The acid-base
balance or pH of the body fluids is maintained by a closely
regulated mechanism. This involves the body buffers,
the respiratory system and the kidney. Some common
definitions are given in Box 29.1. Functions of hydrogen
ions include:
1. The gradient of H+ concentration between inner and
outer mitochondrial membrane acts as the driving
force for oxidative phosphorylation.
2. The surface charge and physical configuration of proteins

are affected by changes in hydrogen ion concentration.
3. Hydrogen ion concentration decides the ionization of
weak acids and thus affects their physiological functions.

¾¾Renal regulation of pH
¾¾Relation of pH and potassium
¾¾Respiratory acidosis
¾¾Metabolic acidosis
¾¾Respiratory alkalosis
¾¾Metabolic alkalosis

ACIDS AND BASES
Definition
The electrolyte theory of dissociation was proposed by
Arrhenius (Nobel prize, 1903). According to the definition
proposed by Bronsted, acids are substances that are

SPL
Sorensen
1868–1939

Svante Arrhenius
NP 1903
1859–1927

Johannes N
Bronsted
1879–1947

Chapter 29:   Acid-Base Balance and pH
capable of donating protons and bases are those that
accept protons. Acids are proton donors and bases are
proton acceptors. A few examples are shown below:
Acids

Bases

HA

H + A NH3 + H+

HCl

H+ + Cl –HCO3– +H+

+

H2CO3

–

NH4+
H2CO3

H+ + HCO3–

Weak and Strong Acids
i. The extent of dissociation decides whether they are strong
acids or weak acids. Strong acids dissociate completely

in solution, while weak acids ionize incompletely, for
example,
HCl
H+ + Cl– (Complete)
H2CO3

H+ + HCO3– (Partial)

ii.In a solution of HCl, almost all the molecules
dissociate and exist as H+ and Cl– ions. Hence the
concentration of H+ is very high and it is a strong acid.
iii. But in the case of a weak acid (e.g. acetic acid), it will
ionize only partially. So, the number of acid molecules
existing in the ionized state is much less, may be only
50%.

Dissociation Constant
i. Since the dissociation of an acid is a freely reversible
reaction, at equilibrium the ratio between dissociated
and undissociated particle is a constant. The dissociation
constant (Ka) of an acid is given by the formula,
Ka =

[H + ] [A − ]
[HA]

Box 29.1: Terms explained
Term
pH
Acids

Bases
Strong acids
Weak acids
pK value
Alkali reserve
Buffers

Definition and explanations
Negative logarithm of hydrogen ion concentration. Normal value 7.4 (range 7.38–7.42)
Proton donors; pH <7
Proton acceptors; pH > 7
Acids which ionize completely; e.g. HCl
Acids which ionize incompletely, e.g. H2CO3
pH at which the acid is half ionised; Salt : Acid
=1:1
Bicarbonate available to neutralise acids;
Normal 24 mmol/L (range 22–26 mmol/L)
Solutions minimize changes in pH

391

Where [H+] is the concentration of hydrogen ions, [A–]
= the concentration of anions or conjugate base, and
[HA] is the concentration of undissociated molecules.
ii. The pH at which the acid is half ionized is called pKa
of an acid which is constant at a particular temperature
and pressure.
iii. Strong acids will have a low pKa and weak acids have
a higher pKa.

Acidity of a Solution and pH
i. The acidity of a solution is measured by
noting the hydrogen ion concentration
in the solution and obtained by the
equation.
[H+] = Ka

[acid] [HA]
or
[base]
A−

where Ka is the dissociation constant.
ii. To make it easier, Sorensen expressed
the H+ concentration as the negative of
the logarithm (logarithm to the base
10) of hydrogen ion concentration, and
is designated as the pH. Therefore,
pH = –log [H+] = log

1
[H + ]

Lawrence J
Henderson
1878–1942

KA Hasselbalch
1874–1962

iii. Thus the pH value is inversely proportional to the
acidity. Lower the pH, higher the acidity or hydrogen
ion concentration while higher the pH, the acidity is
lower (Table 29.1).
iv. At a pH of 1, the hydrogen ion concentration is 10
times that of a solution with a pH 2 and 100 times
that of a solution with a pH of 3 and so on. The pH
7 indicates the neutral pH, when the hydrogen ion
TABLE 29.1: Relation between hydrogen ions, hydroxyl ions and
pH of aqueous solutions. Ionic product of water = [H+][OH–] =
10–14
[OH–]
mols/liter

[H+]
mols/liter

log
[H+]

–log[H+]
=pH

pOH

Inference

1 × 1013

1 × 101

–1

1

13

Strong acid

1 × 1010

1 × 10–4

–4

4

10

Acid

1 × 107

1 × 10–7

–7

7

7

Neutral

1 × 104

1 × 1010

–10

10

4

Alkali

1 × 10

1 × 10

–13

13

1

Strong alkali

1

13

392 Textbook of Biochemistry
concentration is 100 nanomoles/liter. The pH meter is
described in Chapter 35.

The Effect of Salt Upon the Dissociation
i. The relationship between pH, pKa, concentration of
acid and conjugate base (or salt) is expressed by the
Henderson-Hasselbalch equation,
pH = pKa + log

[base]
[salt]
or pH = pKa + log
[acid]
[acid]

When [base] = [acid]; then pH = pKa
ii.Therefore, when the concentration of base and acid
are the same, then pH is equal to pKa. Thus, when
the acid is half ionized, pH and pKa have the same
values.

BUFFERS
Definition
Buffers are solutions which can resist changes in pH
when acid or alkali is added.

Composition of a Buffer

Buffers are of two types:
a. Mixtures of weak acids with their salt with a strong
base or
b. Mixtures of weak bases with their salt with a strong
acid. A few examples are given below:

i.H2CO3/NaHCO3 (Bicarbonate buffer)
(carbonic acid and sodium bicarbonate)

ii.CH3COOH/CH3COO Na (Acetate buffer)
(acetic acid and sodium acetate)

iii.Na2HPO4/NaH2PO4 (Phosphate buffer)

Factors Affecting pH of a Buffer
The pH of a buffer solution is determined by two factors:
a. The value of pK: The lower the value of pK, the
lower is the pH of the solution.
b.The ratio of salt to acid concentrations: Actual
concen­trations of salt and acid in a buffer solution
may be varying widely, with no change in pH, so long
as the ratio of the concentrations remains the same.

Factors Affecting Buffer Capacity
i. On the other hand, the buffer capacity is determined
by the actual concentrations of salt and acid present,
as well as by their ratio.
ii. Buffering capacity is the number of grams of strong
acid or alkali which is necessary for a change in pH of
one unit of one litre of buffer solution.

iii. The buffering capacity of a buffer is defined as the
ability of the buffer to resist changes in pH when
an acid or base is added.

How do Buffers Act?
i. Buffer solutions consist of mixtures of a weak acid or
base and its salt.
ii. To take an example, when hydrochloric acid is
added to the acetate buffer, the salt reacts with the
acid forming the weak acid, acetic acid and its salt.
Similarly when a base is added, the acid reacts with
it forming salt and water. Thus changes in the pH are
minimized.
CH3–COOH + NaOH → CH3–COONa + H2O
CH3–COONa + HCl → CH3–COOH + NaCl
iii.The buffer capacity is determined by the absolute
concentration of the salt and acid. But the pH of
the buffer is dependent on the relative proportion of
the salt and acid (see the Henderson-Hasselbalch’s
equation).
iv. When the ratio between salt and acid is 10:1, the pH
will be 1 unit higher than the pKa. When the ratio
between salt and acid is 1:10, the pH will be 1 unit
lower than the pKa.

Application of the Equation
i. The pH of a buffer on addition of a known quantity
of acid and alkali can therefore be predicted by the
equation.
ii. Moreover, the concentration of salt or acid can be

found out by measuring the pH.
iii. The Henderson-Hasselbalch’s equation, therefore
has great practical application in clinical practice
in assessing the acid-base status, and predicting the
limits of the compensation of body buffers.


Chapter 29:   Acid-Base Balance and pH

Effective Range of a Buffer
A buffer is most effective when the concentrations of salt
and acid are equal or when pH = pKa. The effective range
of a buffer is 1 pH unit higher or lower than pKa. Since
the pKa values of most of the acids produced in the body
are well below the physiological pH, they immediately
ionize and add H+ to the medium. This would necessitate
effective buffering. Phosphate buffer is effective at a wide
range, because it has 3 pKa values.

ACID-BASE BALANCE
Normal pH
The pH of plasma is 7.4 (average hydrogen ion
concentration of 40 nmol/L). In normal life, the variation
of plasma pH is very small. The pH of plasma is maintained
within a narrow range of 7.38 to 7.42. The pH of the
interstitial fluid is generally 0.5 units below that of the
plasma.

Acidosis
If the pH is below 7.38, it is called acidosis. Life is

threatened when the pH is lowered below 7.25. Acidosis
leads to CNS depression and coma. Death occurs when pH
is below 7.0.

Alkalosis
When the pH is more than 7.42, it is alkalosis. It is very
dangerous if pH is increased above 7.55. Alkalosis induces
neuromuscular hyperexcitability and tetany. Death occurs
when the pH is above 7.6.

Volatile and Fixed Acids
i. During the normal metabolism, the acids produced
may be volatile acids like carbonic acid or nonvolatile
(fixed) acids like lactate, keto acids, sulfuric acid and
phosphoric acid.
ii. The metabolism produces nearly 20,000 milli
equivalents (mEq) of carbonic acid and 60–80 mEq of
fixed acids per day.
iii. The lactate and keto acids are produced in relatively
fixed amounts by normal metabolic activity, e.g. 1
mol of glucose produces 2 mols of lactic acid.

393

iv. The dietary protein content decides the amount of
sulfuric and phosphoric acids. The sulfoproteins yield
sulfuric acid and phospho­proteins and nucleo­proteins
produce phosphoric acid. On an average about 3 g
of phosphoric acid and about 3 g sulfuric acid are
produced per day.

v. The carbonic acid, being volatile, is eliminated as CO2
by the lungs. The fixed acids are buffered and later on
the H+ are excreted by the kidney.

Mechanisms of Regulation of pH
These mechanisms are interrelated. See Box 29.2.

BUFFERS OF THE BODY FLUIDS
Buffers are the first line of defense against acid load. These
buffer systems are enumerated in Table 29.2. The buffers
are effective as long as the acid load is not excessive, and
the alkali reserve is not exhausted. Once the base is utilized
in this reaction, it is to be replenished to meet further
challenge.

Bicarbonate Buffer System
i. The most important buffer system in the plasma is the
bicarbonate-carbonic acid system (NaHCO3/H2CO3).
It accounts for 65% of buffering capacity in plasma
and 40% of buffering action in the whole body.
Box. 29.2: Mechanisms of regulation of pH
First line of defense
Second line of defense
Third line of defense

: Blood buffers
: Respiratory regulation
: Renal regulation

TABLE 29.2: Buffer systems of the body

1.

2.

3.

Extracellular
fluid

Intracellular
fluid

Erythrocyte
fluid

NaHCO3
H2 CO3

K2HPO4
KH2PO4

(bicarbonate)

(phosphate)

K+Hb
H+Hb
(hemoglobin)

Na2HPO4
NaH2PO4

K+Protein
H+Protein

K2HPO4
KH2PO4

(phosphate)

(protein buffer)

(phosphate)

Na+Albumin
H+Albumin

KHCO3
H2CO3

KHCO3
H2CO3


394 Textbook of Biochemistry
ii. The base constituent, bicarbonate (HCO3–), is regulated
by the kidney (metabolic component).
iii. While the acid part, carbonic acid (H2CO3), is under
respiratory regulation (respiratory component).

iv.The normal bicarbonate level of plasma is 24
mmol/L. The normal pCO2 of arterial blood is 40
mm of Hg. The normal carbonic acid concentration
in blood is 1.2 mmol/L. The pKa for carbonic acid
is 6.1. Substituting these values in the HendersonHasselbalch’s equation,
pH = pKa + log
7.4 = 6.1 + log

−
3

[HCO ]
[H 2 CO3 ]

24
1.2

   = 6.1 + log 20 = 6.1 + 1.3
v. Hence, the ratio of HCO3– to H2CO3 at pH 7.4 is 20
under normal conditions. This is much higher than
the theoretical value of 1 which ensures maximum
effectiveness.
vi. The bicarbonate carbonic acid buffer system is the
most important for the following reasons:

a.Presence of bicarbonate in relatively high
concentrations.

b. The components are under physiological control,
CO2 by lungs and bicarbonate by kidneys.

Alkali Reserve
Bicarbonate represents the alkali reserve and it has to
be sufficiently high to meet the acid load. If it was too
low to give a ratio of 1, all the HCO3– would have been
exhausted within a very short time; and buffering will not
be effective. So, under physiological circumstances, the
ratio of 20 (a high alkali reserve) ensures high buffering
efficiency against acids.

Antilog of 0.6 = 4; hence the ratio is 4. This is found to
be true under physiological condition.
The phosphate buffer system is found to be effective
at a wide pH range, because it has more than one ionizable
group and the pKa values are different for both.
= 1.96 H+ + H2PO4–
H3PO4 pKa
→

H2PO4–

pKa = 6.8 H++ HPO4= (Na2HPO4 /NaH2PO4)
→

HPO4=

pKa = 12.4 H+ + PO4º

→

In the body, Na2HPO4/NaH2PO4 is an effective buffer
system, because its pKa value is nearest to physiological pH.

Protein Buffer System
Buffering capacity of protein depends on the pKa value of
ionizable side chains. The most effective group is histidine
imidazole group with a pKa value of 6.1.The role of the
hemoglobin buffer is considered along with the respiratory
regulation of pH.

Relative Capacity of Buffer Systems
In the body, 52% buffer activity is in tissue cells and 6%
in RBCs. Rest 43% is by extracellular buffers. In plasma
and extracellular space, about 40% buffering action is by
bicarbonate system; 1% by proteins and 1% by phosphate
buffer system (Fig. 29.1).

Buffers Act Quickly, But Not Permanently
Buffers can respond immediately to addition of acid or
base, but they do not serve to eliminate the acid from the
body. They are also unable to replenish the alkali reserve of
the body. For the final elimination of acids, the respiratory
and renal regulations are very essential.

Phosphate Buffer System
It is mainly an intracellular buffer. Its concentration in plasma
is very low. The pKa value is 6.8. So applying the equation,


pH (7.4)= pKa (6.8) + log

or 0.6 = log

[salt]
[acid]

[salt]
[acid]
Fig. 29.1: Intracellular buffers play a significant role to combat acid
load of the body


Chapter 29:   Acid-Base Balance and pH

RESPIRATORY REGULATION OF pH
The Second Line of Defense
i. This is achieved by changing the pCO2 (or carbonic
acid, the denominator in the equation). The CO2
diffuses from the cells into the extracellular fluid and
reaches the lungs through the blood.
ii. The rate of respiration (rate of elimination of CO2) is
controlled by the chemoreceptors in the respiratory
center which are sensitive to changes in the pH of blood.
iii. When there is a fall in pH of plasma (acidosis), the
respiratory rate is stimulated resulting in hyperventilation. This would eliminate more CO2, thus lowering
the H2CO3 level (Box 29.3).
iv. However, this can not continue for long. The respiratory
system responds to any change in pH immediately, but

it cannot proceed to completion.

Action of Hemoglobin
i. The hemoglobin serves to transport the CO2 formed in
the tissues, with minimum change in pH (see isohydric
transport, Chapter 22).
ii. Side by side, it serves to generate bicarbonate or alkali
reserve by the activity of the carbonic anhydrase
system (see Chapter 22).

Carbonic anhydrase
CO2 + H2O
H2CO3
H2CO
HCO3-- + H+
+
-H + Hb
HHb
iii. The reverse occurs in the lungs during oxygenation
and elimination of CO2. When the blood reaches
the lungs, the bicarbonate re-enters the erythrocytes
by reversal of chloride shift. It combines with H+
liberated on oxygenation of hemoglobin to form
carbonic acid which dissociates into CO2 and H2O.
CO2 is thus eliminated by the lungs.
HHb + O2
HbO2 + H+
–
+
HCO3 + H

H2CO3
H2CO3
H2O + CO2
iv. The activity of the carbonic anhydrase (also called
carbonate dehydratase) increases in acidosis and
decreases with decrease in H+ concentration.

395

pH is lower than that of extracellular fluid (pH = 7.4). This
is called acidification of urine. The pH of the urine may
vary from as low as 4.5 to as high as 9.8, depending on the
amount of acid excreted. The major renal mechanisms for
regulation of pH are:
A. Excretion of H+ (Fig. 29.2)
B. Reabsorption of bicarbonate (recovery of bicarbonate)
(Fig. 29.3)
C. Excretion of titratable acid (net acid excretion) (Fig.
29.4)
D. Excretion of NH4+ (ammonium ions) (Fig.29.5).

Excretion of H+; Generation of Bicarbonate
i.This process occurs in the proximal convoluted
tubules (Fig. 29.2).
ii. The CO2 combines with water to form carbonic acid,
with the help of carbonic anhydrase. The H2CO3 then
ionizes to H+ and bicarbonate.
iii. The hydrogen ions are secreted into the tubular lumen;
in exchange for Na+ reabsorbed. These Na+ ions along
with HCO3– will be reabsorbed into the blood.

iv. There is net excretion of hydrogen ions, and net
generation of bicarbonate. So this mechanism serves
to increase the alkali reserve.

Reabsorption of Bicarbonate
i. This is mainly a mechanism to conserve base. There is
no net excretion of H+ (Fig. 29.3).
ii. The cells of the PCT have a sodium hydrogen
exchanger. When Na+ enters the cell, hydrogen ions
from the cell are secreted into the luminal fluid. The
hydrogen ions are generated within the cell by the
action of carbonic anhydrase.

RENAL REGULATION OF pH
An important function of the kidney is to regulate the pH of
the extracellular fluid. Normal urine has a pH around 6; this

Fig. 29.2: Excretion of hydrogen ions in the proximal tubules; CA =
Carbonic anhydrase


396 Textbook of Biochemistry
iii. The hydrogen ions secreted into the luminal fluid is
required for the reabsorption of filtered bicarbonate.
iv. Bicarbonate is filtered by the glomerulus. This is
completely reabsorbed by the proximal convoluted
tubule, so that the urine is normally bicarbonate free.
v. The bicarbonate combines with H+ in tubular fluid to
form carbonic acid. It dissociates into water and CO2.
The CO2 diffuses into the cell, which again combines

with water to form carbonic acid.
vi. In the cell, it again ionizes to H+ that is secreted into
lumen in exchange for Na+. The HCO3– is reabsorbed
into plasma along with Na+.
vii. Here, there is no net excretion of H+ or generation
of new bicarbonate. The net effect of these processes
is the reabsorption of filtered bicarbonate which is
Box 29.3: Summary of buffering against acid load
Stages
FeaturesBuffer

components
Normal
Normal raio = 20:1
HCO3– (N)

________


Normal pH = 7.4

First line of defense
Acidosis; H enters
Plasma buffer system
blood, bicarbonate

is used up
Second line defense
Hyperventilation
RespiratoryH2CO3 →H2O +

compensationCO2↑­
+

H2CO3 (N)
HCO3– (↓↓)

H2CO3 (↓)

Partially compen-
sated acidosis

Bicarbonate ↓;
HCO3– (↓↓)
pH ↓¯H2CO3 (↓↓)

Third line of defense
kidney mechanism




Excretion of H+;HCO3– (↓↓)
Reabsorption of
H2CO3 (↓↓)
bicarbonate;
Ratio and pH
tend to restore

Fig. 29.3: Reabsorption of bicarbonate from the tubular fluid; CA =
Carbonic anhydrase

mediated by the Sodium-Hydrogen exchanger. But this
mechanism prevents the loss of bicarbonate through
urine.
+

Excretion of H as Titratable Acid
i. In the distal convoluted tubules net acid excretion
occurs. Hydrogen ions are secreted by the distal
tubules and collecting ducts by hydrogen ion-ATPase
located in the apical cell membrane. The hydrogen
ions are generated in the tubular cell by a reaction
catalyzed by carbonic anhydrase. The bicarbonate
generated within the cell passes into plasma.
ii.The term titratable acidity of urine refers to the
number of milliliters of N/10 NaOH required to titrate
1 liter of urine to pH 7.4. This is a measure of net
acid excretion by the kidney.
iii. The major titratable acid present in the urine is sodium
acid phosphate. As the tubular fluid passes down the
renal tubules more and more H+ are secreted into the
luminal fluid so that its pH steadily falls. The process
starts in the proximal tubules, but continues up to the
distal tubules.
iv. Due to the Na+ to H+ exchange occurring at the renal
tubular cell boarder, the Na2HPO4 (basic phosphate)
is converted to NaH2PO4 (acid phosphate) (Fig. 29.4).
As a result, the pH of tubular fluid falls.
v. The acid and basic phosphate pair is considered as the
urinary buffer. The maximum limit of acidification

is pH 4.5. This process is inhibited by carbonic
anhydrase inhibitors like acetazolamide.

Fig. 29.4: Phosphate mechanism in tubules


Chapter 29:   Acid-Base Balance and pH

Excretion of Ammonium Ions
i. This predominantly occurs at the distal convoluted
tubules. This would help to excrete H+ and reabsorb
HCO3– (Fig. 29.5).
ii.This mechanism also helps to trap hydrogen ions
in the urine, so that large quantity of acid could be
excreted with minor changes in pH. The excretion of
ammonia helps in the elimination of hydrogen ions
without appreciable change in the pH of the urine.
iii.The Glutaminase present in the tubular cells can
hydrolyze glutamine to ammonia and glutamic acid.
The NH3 (ammonia) diffuses into the luminal fluid and
combines with H+ to form NH4+(ammonium ion). The
glutaminase activity is increased in acidosis. So large
quantity of H+ ions are excreted as NH4+ in acidosis.
iv. Since it is a positively charged ion, it can accompany
negatively charged acid anions; so Na+ and K+ are
conserved (Fig. 29.5).
v. Normally, about 70 mEq/L of acid is excreted daily;
but in condition of acidosis, this can rise to 400 mEq/
day.
vi. The enhanced activity of glutaminase and increased

excretion of NH4+ take about 3–4 days to set in under
conditions of acidosis. But once established, it has
high capacity to eliminate acid.
vii. Ammonia is estimated in urine, after addition of
formaldehyde. The titratable acidity plus the ammonia
content will be a measure of acid excreted from

397

the body. Maximum urine acidity reached is 4.4.
A summary of buffering of acid load in the body is
shown in Table 29.3.
CELLULAR BUFFERS
Cytoplasmic pH varies from 6.8 to 7.3. Intracellular pH modulates a
variety of cell functions:
1. The activity of several enzymes is sensitive to changes in pH.
2. Reduction in pH reduces the contractility of actin and myosin in
muscles.
3. The electrical properties of excitable cells are also affected by
changes in pH.
Intracellular buffers are depicted in Figure 29.1. The major tissues
involved in cellular buffering are bone and skeletal muscle. The
buffering of acid is achieved by the exchange of H+ that enters into
the cells for Na+ or K+ ions.

Relationship of pH with K+ Ion Balance
i. When there is increase in H+ in extracellular fluid
(ECF), there may be exchange of H+ with K+ from
within the cells. Net effect is an apparent increase in
ECF potassium level (hyperkalemia).

ii.In general, acute acidosis is associated with
hyperkalemia and acute alkalosis with hypokalemia.
iii. However, in renal tubular acidosis, due to failure to
excrete hydrogen ions, potassium is lost in urine; then
hypokalemia results.
iv. Sudden hypokalemia may develop during the correction
of acidosis. K+ may go back into the cells, suddenly
lowering the plasma K+. Hence it is important to
maintain the K+ balance during correction of alkalosis.

Factors affecting Renal Acid Excretion







1. Increased filtered load of bicarbonate
2. Decrease in ECF volume
3. Decrease in plasma pH
4. Increase in pCO2 of blood
5.Hypokalemia
6. Aldosterone secretion.

DISTURBANCES IN ACID-BASE BALANCE

Fig. 29.5: Ammonia mechanism

Acidosis is the clinical state, where acids accumulate or

bases are lost. A loss of acid or accumulation of base leads
to alkalosis. The body cells can tolerate only a narrow
range of pH. The extreme ranges of pH are between 7.0 and


398 Textbook of Biochemistry
7.6, beyond which life is not possible. Box 29.4 shows the
conditions in which acid-base parameters are to be checked.
Box 29.5 shows the steps to the clinical assessment of acid
base status. Box 29.6 summarizes the abnormal findings.

Classification of Acid-Base Disturbances
Acidosis (fall in pH)
a. Respiratory acidosis: Primary excess of carbonic acid.
b. Metabolic acidosis: Primary deficit of bicarbonate
(Box 29.6).

Alkalosis (rise in pH)
a. Respiratory alkalosis: Primary deficit of carbonic
acid.
b. Metabolic alkalosis: Primary excess of bicarbonate
(Box 29.6).

Compensatory Responses
Each of the above disturbance will be followed by a
secondary compensatory change in the counteracting
variable, e.g. a primary change in bicarbonate involves
an alteration in pCO2. Depending on the extent of the
compensatory change there are different stages (Table
29.3). In actual clinical states, patients will have different

states of compen­sation (Box 29.7). The compensatory
(adaptive) responses are:
a. A primary change in bicarbonate involves an alteration
in pCO2. The direction of the change is the same as the
primary change and there is an attempt at restoring the
ratio to 20 and pH to 7.4.
b. Adaptive response is always in the same direc­tion as
the primary disturbance. Primary decrease in arterial
bicarbonate involves a reduction in arterial blood
pCO2 by alveolar hyperventilation.

Box 29.4: Acid-base parameters are to be checked in patents with

Box 29.6. Acid-base disturbances

1.
2.
3.
4.
5.
6.

pCO2
pCO2
HCO3
HCO3
H+
H+

Any serious illness

Multi organ failure
Respiratory failure
Cardiac failure
Uncontrolled diabetes mellitus
Poisoning by barbiturates and ethylene glycol

Box 29.5: Steps to the clinical assessment of acid-base disturbances

> 45 mm Hg =
< 35 mm Hg =
> 33 mmol/L =
< 22 mmol/L =
> 45 nmol/L =
< 35 nmol/L =

Respiratory acidosis
Respiratory alkalosis
Metabolic alkalosis
Metabolic acidosis
Acidosis
Alkalosis

Box 29.7. Acid base disturbances. Expected renal and respiratory compensations

1. Assess pH (normal 7.4); pH <7.35 is acidemia and >7.45 is
alkalemia
2. Serum bicarbonate level: See Box 29.6.
3. Assess arterial pCO2: See Box 29.6.

4.

Check compensatory response: Compensation never
overcompensates the pH. If pH is <7.4, acidosis is the primary
disorder. If pH is >7.4, alkalosis is primary.
5. Assess anion gap.
6. Assess the change in serum anion gap/change in bicarbonate.
7. Assess if there is any underlying cause.

Metabolic acidosis: Expect pCO2 to be reduced by 1 mm Hg
for every 1 mmol/L drop in bicarbonate.
Metabolic alkalosis: Expect pCO2 to be increased by 0.6 mm
Hg for every 1 mmol/L rise in bicarbonate.
Acute respiratory acidosis: Expect 1 mmol/L increase in
bicarbonate per 10 mm Hg rise in pCO2.
Chronic respiratory acidosis: Expect 3.5 mmol/L increase in
bicarbonate per 10 mm Hg rise in pCO2.
Acute respiratory alkalosis: Expect 2 mmol/L decrease in
bicarbonate per 10 mm Hg fall in pCO2.
Chronic respiratory alkalosis: Expect 4 mmol/L decrease in
bicarbonate per 10 mm Hg fall in pCO2.

TABLE 29.3: Types of acid-base disturbances

Disturbance

pH

Primary change

Ratio

Secondary change

Metabolic acidosis

Decreased

Deficit of bicarbonate

<20

Decrease in PaCO2

Metabolic alkalosis

Increased

Excess of bicarbonate

>20

Increase in PaCO2

Respiratory acidosis

Decreased

Excess of carbonic acid

<20

Increase in bicarbonate

Respiratory alkalosis

Increased

Deficit of carbonic acid

>20

Decrease in bicarbonate


Chapter 29:   Acid-Base Balance and pH
c. Similarly, a primary increase in arterial pCO2
involves an increase in arterial bicarbonate by an
increase in bicarbonate reabsorption by the kidney.
d. The compensatory change will try to restore the pH
to normal. However, the compensatory change cannot
fully correct a disturbance.
e. Clinically, acid-base disturbance states may be
divided into:

i. Uncompensated

ii. Partially compensated

iii. Fully compensated (Table 29.4).

Mixed Responses

i. If the disturbance is pure, it is not difficult to accurately
assess the nature of the disturbance (Box 29.7). In
mixed disturbances, both HCO3– and H2CO3 levels are
altered (Fig. 29.6).
ii. The adaptive response always involves a change in
the counteracting variable; e.g. a primary change in
bicarbonate involves an alteration in pCO2.
iii. Adaptive response is always in the same direction as
the primary disturbance.
iv. Depending on the extent of the compensatory change
there are different stages. Looking at the parameters,
the stage of the compensation can be identified
(Table 29.4).

Chemical Pathology of Acid-Base Disturbances
Metabolic Acidosis
i. It is due to a primary deficit in the bicarbonate. This
may result from an accumulation of acid or depletion
of bicarbonate.
ii. When there is excess acid production, the bicarbonate
is used up for buffering. Depending on the cause, the
anion gap is altered.

Anion Gap
i. The sum of cations and anions in ECF is always equal,
so as to maintain the electrical neutrality. Sodium and
potassium together account for 95% of the cations
whereas chloride and bicarbonate account for only
86% of the anions (Fig. 29.7). Only these electrolytes
are commonly measured.

ii. Hence, there is always a difference between the
measured cations and the anions. The unmeasured
anions constitute the anion gap. This is due to the
presence of protein anions, sulphate, phosphate and
organic acids.
iii. The anion gap is calculated as the difference between
(Na+ + K+) and (HCO3– + Cl–). Normally this is about
12 mmol/L.

TABLE 29.4: Stages of compensation
Stage

pH

HCO3

PaCO2

Ratio

Metabolic acidosis

Low

Low

N

<20

 Uncompensated

Low

Low

N

<20

  Partially compensated

Low

Low

Low

<20

  Fully compensated

N

Low

Low

Metabolic alkalosis

High

High

N

>20

 Uncompensated

High

High

N

>20

  Partially compensated

High

High

High

>20

  Fully compensated

N

High

High

20

Respiratory acidosis

Low

N

High

<20

 Uncompensated

Low

N

High

<20

  Partially compensated

Low

High

High

<20

  Fully compensated

N

High

High

20

Respiratory alkalosis

High

N

Low

>20

 Uncompensated

High

N

Low

>20

  Partially compensated

High

Low

Low

>20

  Fully compensated

N

Low

Low

20

399

20

Fig. 29.6: Bicarbonate diagram


400 Textbook of Biochemistry
High Anion Gap Metabolic Acidosis (HAGMA)

Normal Anion Gap Metabolic Acidosis (NAGMA)

i. A value between 15 and 20 is accepted as reliable
index of accumulation of acid anions in metabolic
acidosis (HAGMA) (Table 29.5).
ii. Renal failure: The excretion of H+ as well as generation
of bicarbonate are both deficient. The anion gap
increases due to accumulation of other buffer anions.
iii. Diabetic ketoacidosis (see Chapter 12).
iv. Lactic acidosis: Normal lactic acid content in plasma
is less than 2 mmol/L. It is increased in tissue hypoxia,
circulatory failure, and intake of biguanides (Box
29.8). Lactic acidosis causes a raised anion gap (Box
29.8), whereas diarrhea causes a normal anion gap
acidosis (Table 29.6).
Suppose 5 mmol/L lactic acid has entered in blood; this is buffered

When there is a loss of both anions and cations, the
anion gap is normal, but acidosis may prevail. Causes are
described in Table 29.6.
i. Diarrhea: Loss of intestinal secretions lead to
acidosis. Bicarbonate, sodium and potassium are lost.

ii. Hyperchloremic acidosis may occur in renal
tubular acidosis, acetazolamide (carbonic anhydrase
inhibitor) therapy, and ureteric transplantation into
large gut (done for bladder carcinoma).

by bicarbonate, resulting in 5 mmol/L of sodium lactate and 5
mmol/L of carbonic acid. The carbonic acid is dissociated into
water and carbon dioxide, which is removed by lung ventillation.
The result is lowering of bicarbonate by 5 mmol and presence of
5 mmol of unmeasured anion (lactate), with no changes in sodium
or chloride. So, anion gap is increased. In contrast, diarrhea
results in the loss of bicarbonate. NaCl is reabsorbed more from
kidney tubules to maintain the extracellular volume, resulting in
the increase in serum chloride. This chloride compensates for the
fall in bicarbonate. So, diarrhea results in hyperchloremic, normal
anion gap, metabolic acidosis.

v. The gap may be apparently narrowed when cations
are decreased (K, Mg and Ca) or when there is
hypoalbuminemia. Similarly a spurious elevation is
seen in hypergamma globulinemia when positively
charged proteins are elevated or when cations are
increased (K, Ca and Mg) or in alkalosis when negative
charges on albumin are increased.

Fig. 29.7: Gamblegram showing cations on the left and anions on

the right side. Such bar diagrams were first depicted by Gamble,
hence these are called Gamble grams

Box 29.8. Types of lactic acidosis
Type A :




Type B:





Impaired lactic acid production with hypoxia.
It is seen in Tissue hypoxia (anaerobic metabolism);
Shock (anaphylactic, septic, cardiac);
Lung hypoxia, Carbon monoxide poisoning,
seizures
Impaired lactic acid metabolism without hypoxia.
It is seen in Liver dysfunctions (toxins, alcohol, inborn errors);
Mitochondrial disorders (less oxidative phosphory­
lation and more anaerobic glycolysis)
Thiamine deficiency (defective pyruvate dehydro­
genase)

TABLE 29.5: High anion gap metabolic acidosis (HAGMA)
(organic acidosis)
Cause

Remarks

Renal failure

Sulfuric, phosphoric, organic anions. Decreased
ammonium ion formation. Na+/H+ exchange
results in decreased acid excretion

Ketosis

Acetoacetate; beta hydroxy butyrate anions. Seen
in diabetes mellitus or starvation

Lactic
acidosis

Lactate anion. It accumulates when the rate of
production exceeds the rate of consumption

Salicylate

Aspirin poisoning

Amino
acidurias

Acidic metabolic intermediates
Accumulation due to block in the normal metabolic
pathway

Organic
acidurias

Organic acids (methyl malonic acid, propionic
acid, etc.) excreted

Methanol

Formate, Glycolate, Oxalate ions. Acids formed lead
to increase in AG. Increase in plasma osmolality.
Osmolal gap is also seen

Drugs

Corticosteroids, Dimercaprol, Ethacrynic acid,
Furosemide, Methanol, Nitrates, Salicylates, Thiazides


Chapter 29:   Acid-Base Balance and pH


a. Renal tubular acidosis may be due to failure to
excrete acid or reabsorb bicarbonate.

b. Chloride is elevated since electrical neutrality has
to be maintained.

c. In ureteric transplantation, the chloride ions are
reabsorbed in exchange for bicarbonate ions lost,
leading to hyperchloremic acidosis.

d.Acetazolamide therapy results in metabolic

acidosis because HCO3– generation and H+
secretion are affected.
iii. Urine anion gap (UAG) is useful to estimate the
ammonium excretion. It is calculated as UAG = UNa
+ UK – UCl
The normal value is –20 to –50 mmol/L. In metabolic
acidosis, the NH4Cl excretion increases, and UAG
becomes –75 or more. But in RTA, ammonium excretion is
defective, and UAG has positive value. Causes for RTA are
enumerated in Box 29.9.

Decreased Anion Gap is seen in
¾¾
¾¾
¾¾
¾¾

Hypoalbuminemia
Multiple myeloma (paraproteinemia)
Bromide intoxication
Hypercalcemia

Serum Albumin Levels and Anion Gap
Normal anion gap is affected by the patient’s serum albumin level: As a
general rule of thumb, the normal anion gap is roughly three times the
albumin value, e.g for a patient with an albumin of 4.0, the normal anion
gap would be 12. For a patient with chronic liver disease and an albumin
of 2.0, the upper limit of normal for the anion gap would be 6. The ceiling

401

value for a normal anion gap is reduced by 2.5 for every 1g/dL reduction
in the plasma albumin concentration.

Does the anion gap explain the change in bicarbonate? ∆ anion
gap (Anion gap –12) ~ ∆ [HCO3]. If ∆ anion gap is greater; consider
additional metabolic alkalosis. If ∆ anion gap is less; consider a nonanion gap metabolic acidosis.

Corrected Anion gap is given by the formula Calculated AG + 2.5
(Normal albumin g/dL–Observed albumin in g/dL)

Osmolal Gap
This is the difference between the measured plasma
osmolality and the calculated osmolality, which may be
calculated as
2 × [Na] + [glucose] + [urea]

Box 29.9: Causes of renal tubular acidosis
Type I (Proximal RTA)

Multiple myeloma, amyloidosis

Heavy metals; lead, mercury

Wilson’s disease
Galactosemia
Hyperparathyroidism

Paroxysmal nocturnal hemoglobinuria
Acetazolamide

Type II (Distal RTA)

Autoimmune disorders; SLE, rheumatoid
Hypercalciuria

Amphotericin B, Lithium

Obstructive uropathy

Marfan’s syndrome
Type IV



Impaired aldosterone function

TABLE 29.6: Normal anion gap metabolic acidosis (NAGMA) (inorganic acidosis)
Cause

Remarks

Diarrhea, intestinal fistula

Loss of bicarbonate and cations. Sodium or Potassium or both

RTA Type I

Defective acidification of urine
I or distal RTA, urine pH is >5.5 with hypokalemia
Due to inability to reabsorb bicarbonate

Compensatory increase in chloride (hyperchloremic acidosis)

Type II

II or proximal RTA, urine pH is <5.5, K normal
Due to inability to excrete hydrogen ions

Type IV

Resistance to aldosterone, urine pH <5.5, hyperkalemia

Carbonic anhydrase inhibitors

Loss of bicarbonate, Na and K
Similar to proximal RTA

Ureterosigmioidostomy

Loss of bicarbonate and reabsorption of chloride. Hyperchloremic acidosis

Drugs

Antacids containing magnesium, chlorpropamide, iodide (absorbed from dressings), lithium, polymixin B


402 Textbook of Biochemistry
The normal osmolal gap is <10 mOsm. A high osmolal
gap (> 25) implies the presence of unmeasured osmoles
such as alcohol, methanol, ethylene glycol, etc. Acute
poisoning should be considered in patients with a raised

anion gap metabolic acidosis and an increased plasma
osmolal gap. Poisoning with methanol and ethylene glycol
should be considered. They are metabolized to formic acid
and oxalic acids correspondingly. Methanol will produce
blindness. Ethylene glycol will lead to oxalate crystalluria
and renal failure.

Compensated Metabolic Acidosis
i. Decrease in pH in metabolic acidosis stimulates the
respiratory compensatory mechanism and produces
hyperventilation-Kussmaul respiration to eliminate
carbon dioxide leading to hypocapnia (hypocarbia).
The pCO2 falls and this would attempt to restore the
ratio towards 20 (partial compensation).
ii. Renal compensation: Increased excretion of acid and
conservation of base occurs. Na-H exchange, NH4+
excretion and bicarbonate reabsorption are increased.
As much as 500 mmol acid is excreted per day. The
reabsorption of more bicarbonate also helps to restore
the ratio to 20.
iii. Renal compensation sets in within 2 to 4 days. If the
ratio is restored to 20, the condition is said to be fully
compensated. But unless the cause is also corrected,
restoration of normalcy cannot occur.
iv.Associated hyperkalemia is commonly seen due to
a redistribution of K+ and H+. The intracellular K+
comes out in exchange for H+ moving into the cells.
Hence, care should be taken while correcting acidosis
which may lead to sudden hypokalemia. This is more
likely to happen in treating diabetic ketoacidosis by

giving glucose and insulin together.
v. However changes in albumin level or changes in the
negative charge on the protein molecules can give altered
Anion Gap (AG) values. Therefore when pH increases
the AG may show an increase and in hypoalbuminemia
AG will show a decrease. In order to overcome these
difficulties, a new term “Strong ion gap” (SIG) has been
introduced, which is the corrected AG.

Clinical Features of Metabolic Acidosis
The respiratory response to metabolic acidosis is to
hyperventilate. So there is marked increase in respiratory

rate and in depth of respiration; this is called as Kussmaul
respiration. The acidosis is said to be dangerous when
pH is < 7.2 and serum bicarbonate is <10 mmol/L. In such
conditions, there is depressed myocardial contractility.

Treatment of Metabolic Acidosis
Treatment is to stop the production of acid by giving IV
fluids and insulin. Oxygen is given to patients with lactic
acidosis. In all cases, potassium status to be monitored
closely and promptly corrected.
Bicarbonate requirement: The amount of bicarbonate
required to treat acidosis is calculated from the base deficit.
In cases of acidosis, mEq of base needed = body wt in Kg ×
0.2 – base excess in mEq/L.

Metabolic Alkalosis
i. Primary excess of bicarbonate is the characteristic

feature. Alkalosis occurs when a) excess base is
added, b) base excretion is defective or c) acid is lost.
All these will lead to an excess of bicarbonate, so that
the ratio becomes more than 20. Important causes and
findings are given in Table 29.7. This results either
from the loss of acid or from the gain in base.
ii.Loss of acid may result from severe vomiting or
gastric aspiration leading to loss of chloride and acid.
Therefore, hypochloremic alkalosis results.
iii. Hyperaldosteronism causes retention of sodium and
loss of potassium.
iv.Hypokalemia is closely related to metabolic alkalosis.
In alkalosis, there is an attempt to conserve hydrogen
ions by kidney in exchange for K+. This potassium
loss can lead to hypokalemia.
v. Potassium from ECF will enter the cells in exchange
for H+. So, in alkalosis, pH of urine remains acidic;
hence this is called paradoxic acidosis.

Subclassification of Metabolic Alkalosis
i. In Chloride responsive conditions, urinary chloride is
less than 10 mmol/L. It is seen in prolonged vomiting,
nasogastric aspiration or administration of diuretics.
ii.In Chloride resistant condition, urine chloride is
greater than 10 mmol/L; it is seen in hypertension,
hyperaldosteronism, severe potassium depletion and
Cushing’s syndrome.
iii. Due to the exogenous base which is often iatrogenic.